RNA-Oligonucleotide Quantification Technique for the Enumeration of Uncultivated Bacterial Species

Abstract

Methods for RNA-Oligonucleotide Quantification Technique for the Enumeration of Uncultivated Bacterial Species are disclosed.

1 . A method for identifying and quantifying one or more microorganisms in a sample from an individual, wherein the method comprises:
 a) extracting the total nucleic acid (TNA) from the sample;   b) contacting TNA from the sample with one or more probes that correlate to a microorganism to be quantified, under conditions suitable for hybridization to thereby form a complex; and   c) detecting the amount of the complex,
 wherein the amount of the complex correlates with the amount of TNA of one or more microorganism in the sample. 
   
     
     
         2 . The method of  claim 1 , wherein cultivated microorganisms, uncultivated microorganisms, or both are identified and quantified. 
     
     
         3 . The method of  claim 1 , wherein a plurality of microorganisms are quantified. 
     
     
         4 . The method of  claim 2 , wherein detecting nucleic acid of the microorganism includes detecting 16S rRNA of the microorganism. 
     
     
         5 . The method of  claim 1 , wherein the sample from the individual is obtained from the group consisting of the oral cavity, sinus, esophagus, respiratory tract, lungs, sputum, pharynx, eustachian tube, middle ear, vagina, blood, pus, spinal fluid, and gastrointestinal tract. 
     
     
         6 . The method of  claim 1 , further comprising labeling the nucleic acid molecules of the sample with a detectable label. 
     
     
         7 . The method of  claim 6 , wherein the detectable label is selected from the group consisting of digoxigenin, fluorescent dyes, streptavidin conjugate, magnetic beads, dendrimers, radiolabels, enzymes, colorimetric labels, nanoparticles, and nanocrystals. 
     
     
         8 . The method of  claim 1 , wherein the nucleic acid are bound to a solid support. 
     
     
         9 . The method of  claim 8 , wherein the solid support is selected from the group consisting of nylon membrane, glass, silica chips, polymer, plastic, ceramic, metal, and optical fiber. 
     
     
         10 . A method for assisting in the diagnosis an individual having a disease or condition, the method comprises:
 a) determining the amount TNA of one or more cultivated or uncultivated microorganisms in a sample from the individual,
 wherein an amount of TNA of a microorganism correlates to a disease state or a healthy state. 
   
     
     
         11 . The method of  claim 10 , wherein the amount of TNA between about 10 3  and about 10 6  of a given taxon correlates to a disease state or a healthy state. 
     
     
         12 . A method for diagnosing an individual having a periodontal disease, the method comprises:
 a) determining an amount of TNA of one or more cultivated or uncultivated microorganisms in a sample from the individual,
 wherein the amount of TNA between about 10 3  and about 10 6  of one or more of microorganism correlates to a disease state or a healthy state, and an amount less than 10 3  does not correlate to a disease state or a healthy state. 
   
     
     
         13 . The method of  claim 12 , wherein a periodontal disease state is associated with the amount of TNA between about 10 3  and about 10 6  of  Fusobacterium nucleatum . ss  polymorphum, Actinomyces gerencseriae, Mitsuokella  sp OT — 131,  Prevotella  sp OT 306,  Porphyromonas gingivalis, Peptostreptococcus  strains BS044 or CK035,  Desulfobulbus  sp OT 041 , Synergistetes _OT — 359,  Selenomonas _OT — 134 — 442, TM7_OT — 346 — 349,  Capnocytophaga _OT — 335 , Haemophilus _OT — 035 , Synergistetes _OT — 363 — 453 — 452,  Treponema _OT — 256 — 508 — 517,  Actinomyces _OT — 177, and  Bacteroidetes _OT — 274. 
     
     
         14 . A method of monitoring treatment for a disease associated with one or more bacteria; the method comprises:
 a) determining the amount of TNA of one or more cultivated or uncultivated microorganisms in a sample from the individual at one or more time points,   b) comparing the amount of TNA of the one or more cultivated or uncultivated microorganism from step a) to a standard;
 wherein the amount of TNA between about 10 3  and about 10 6  of one or more of microorganism correlates to the amount of the one or more microorganism present in the sample; and 
 a decrease in bacteria associated with disease indicates successful treatment and an increase in bacterial associated with said disease indicates unsuccessful treatment. 
   
     
     
         15 . A method of determining the efficacy of an oral composition; the method comprises:
 a) contacting the oral composition with a sample from an individual;   b) determining the amount of TNA of one or more cultivated or uncultivated microorganisms in a sample from the individual before and after step a); and   c) comparing the amount of TNA of the one or more cultivated or uncultivated microorganism obtained from step b);
 wherein the amount of TNA between about 10 3  and about 10 6  of one or more of microorganism correlates to the amount of the microorganism in the sample; and a decrease in the amount of TNA in the sample indicates an effective oral composition; and an increase or no change in the amount of TNA in the sample indicates an ineffective oral composition. 
   
     
     
         16 . A method for identifying one or more microorganisms associated with a disease or condition in an individual, the method comprises:
 a) determining the amount TNA of one or more cultivated or uncultivated microorganisms in a sample from the individual,   wherein the amount of the complex correlates with the amount of TNA of one or more microorganism in the sample.   
     
     
         17 . The method of  claim 16 , wherein the amount of TNA that correlates with one or more microorganisms is present in an amount between about 10 3  and about 10 6  in the sample.